The objective is to elucidate the mechanisms of regulation and induction of cytochrome P-450-catalyzed cholesterol 7Alpha-hydroxylase, a rate-limiting enzyme in bile acid metabolism in the liver. A defect in this regulatory enzyme activity may lead to metabolic disorders, such as hypercholesterolemia, atherosclerosis, cholelithiasis and obesity. A specific cytochrome P-450 isozyme which is active in catalyzing 7Alpha-hydroxylation of cholesterol in a reconstituted enzyme system, has been purified from cholestyramine-treated rat liver microsomes. The induction mechanism will be studied using immunoquantitation and in vitro translation system to measure the rate of turnover, synthesis, and the level of mRNA coding for cholesterol 7Alpha-hydroxylase under various factors, such as sex, inducer and diurnal rhythm. Molecular cloning techniques will be approaches to obtain a cDNA for cholesterol 7Alpha-hydroxylase mRNA, which will be used to study the translational regulation of enzyme activity in the liver. Cultured hepatocytes will be isolated from the livers of rats to study the regulation of enzyme activity by the feedback inhibitor, bile acids. The short-term, post-translational modulation of enzyme activity by reversible phosphorylation/dephosphorylation mechanism will be studied in the hepatocytes. The regulation of the cholesterol 7Alpha-hydroxylase activity in genetically obese Zucker rats will also be studied to understand the role of this enzyme in hypercholesterolemia and obesity. Our long-range goal is to understand the mechanisms of regulation of cholesterol 7Alpha-hydroxylase activity and gene expression of this physiologically important enzyme in the liver and its relationship to metabolic diseases.